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Apa protein
The protein encoded by the Rv1860 gene of Mycobacterium tuberculosis (MTB) is called APA protein, due to high abundance of amino acids alanine-proline-alanine (APA) repeats at the C-terminus of the protein. It is a 325 amino acid long, secreted, glycosylated antigen. Recombinant forms of the protein have been used to study its role in pathogenesis of MTB and as a vaccine candidate (1). Significance Tuberculosis is a bacterial infectious disease caused by MTB. Drug resistance has been a big problem with the present vaccine having a highly variable efficacy (2). A strong adaptive immune response has been shown to be vital for protection against tuberculosis (3). Hence, this has been used as a criteria for development of vaccines. Recombinant DNA technology has been a very useful tool in the search for MTB antigens that could provide a strong adaptive immune response, and thus be a good vaccine candidate (4, 5). Amara et al., initially created a MTB genomic expression library to identify immunodominant antigens that react with tuberculosis patient sera (6). Apa protein was one of the antigens identified from this library and further studied. It was cloned, expressed and purified to study its effectiveness as ability to stimulate a protective adaptive immune response in vitro using peripher (1). Strategy - A truncated Rv1860 gene lacking 93 nucleotides at the N-terminus after the start codon, was isolated using polymerase chain reaction (PCR) from the gene expression library for MTB with EcoRI overhang and a Klenow-filled BamHI site. - This gene insert was ligated into the multiple cloning site of the pRSET C vector between the EcoRI and Klenow-filled HindIII restriction sites. The pRSET C vector has a ampicillin resistance gene for selection of plasmid and a sequence coding for six histidine residues upstream of the inserted Rv1860 gene for purificatio n of the protein post-expression (Figure 1). The recombinant plasmids were selected using E. coli DH5alpha strain using ampicillin after transformation. - The recombinant vector was introduced into BL21 (DE3) expression system to express the recombinant protein. This expression sytem has an extra system in the form of DE3 for tightly regulating expression of target gene (Figure 2). The target APA gene is transcribed by T7 RNA polymerase. The expression of T7 RNa polymerase is in the DE3 system and is regulated by a lac operon. In the absence of lactose, a lac repressor, prevents transcription of T7 RNA polymerase. In the presence of lactose, a lactose metabolite called allolactose is made which displaces the lac repressor and induces transcription of lac operon producing T7 RNA polymerase. A synthetic compound, Isopropyl β-D-1-thiogalactopyranoside (IPTG) can substitute for allolactose and cause the same result of producing T7 RNA polymerase and thus express high levels of the target protein (APA here). - A Nickel-nitrilotriacetic acid (Ni-NTA) column (affinity chromatography) was used to purify the expressed protein. The six histidine tag on the N-terminus of the protein binds the Ni-NTA column with the remaning contaminants move out as flow through. Bound protein was eluted using imidazole as this displaces the histidine-tagged APA proten and itself binds Ni-NTA more tightly. - Thus, the pure APA protein was obtained and used for immunoogical studies. References 1. Kumar P, Amara RR, Challu VK, Chadda VK, Satchidanandam V. The Apa protein of Mycobacterium tuberculosis stimulates gamma interferon-secreting CD4+ and CD8+ T cells from purified protein derivative-positive individuals and affords protection in a guinea pig model. Infection and immunity. 2003;71(4):1929-37. Epub 2003/03/26. http://www.ncbi.nlm.nih.gov/pubmed/12654810 PubMed. 2. McShane H. Tuberculosis vaccines: beyond bacille Calmette-Guerin. Philosophical transactions of the Royal Society of London Series B, Biological sciences. 2011;366(1579):2782-9. Epub 2011/09/07. http://www.ncbi.nlm.nih.gov/pubmed/21893541 PubMed. 3. Mackaness GB. The immunology of antituberculous immunity. The American review of respiratory disease. 1968;97(3):337-44. Epub 1968/03/01. http://www.ncbi.nlm.nih.gov/pubmed/4966267 PubMed. 4. Husson RN, Young RA. Genes for the major protein antigens of Mycobacterium tuberculosis: the etiologic agents of tuberculosis and leprosy share an immunodominant antigen. Proceedings of the National Academy of Sciences of the United States of America. 1987;84(6):1679-83. Epub 1987/03/01. http://www.ncbi.nlm.nih.gov/pubmed/3104901 PubMed. 5. Shinnick TM, Krat C, Schadow S. Isolation and restriction site maps of the genes encoding five Mycobacterium tuberculosis proteins. Infection and immunity. 1987;55(7):1718-21. Epub 1987/07/01. http://www.ncbi.nlm.nih.gov/pubmed/3036711 PubMed. 6. Amara RR, Satchidanandam V. Analysis of a genomic DNA expression library of Mycobacterium tuberculosis using tuberculosis patient sera: evidence for modulation of host immune response. Infection and immunity. 1996;64(9):3765-71. Epub 1996/09/01. http://www.ncbi.nlm.nih.gov/pubmed/8751927 PubMed.